LH-RH (luteinizing hormone-releasing hormone) is a peptide consisting of 10 amino acids which was first isolated from the hypothalami of pigs [H. Matsuo et al., Biochem. Biophys. Res. Commun., 43, 1334-1339 (1971)], and has activity to act on the anterior lobes of pituitaries to allow them to release gonadotropic hormones [LH and follicle stimulating hormone (FSH)] [R. N. Clayton et al., Endocr. Rev., 2, 186-209 (1981)]. Of the two kinds of gonadotropic hormones, both coexisting with each other induce secretion of estrogenic hormone for females, and LH alone induces secretion of androgenic hormone for males. As described above, LH-RH is a leading hormone playing an important role in the control of the gonadal and sex hormones, and has affects on the genital organs such as the gonad and the uterus. In particular, drugs for inhibiting the activity of LH-RH are known to stop the progress of prostatic cancer dependent on androgenic hormone. Further, similar drugs are also known to be effective against endometriosis due to hetrotopic abnormal proliferation of endometrial tissue. However, since such drugs are peptides and oral administration thereof is impossible, injection thereof is necessary.
On the other hand, LH-RH receptors exist in LH secretory cells and FSH secretory cells of the anterior lobes of pituitaries, the stimulation of LH-RH is transmitted to these cells through LH-RH receptors. Further, the LH-RH receptors are also known to be expressed in the placenta [A. J. Currie et al., Biochem. Biophys. Res. Commun., 99, 332-338 (1981), Nature, 282, 90-92 (1979), and R. N. Clayton et al., Proc. Natl. Acad. Sci. USA, (1980)], the adrenal medulla [D. R. Pieper et al., Endocrinology, 108, 1148-1155 (1981)], the brain [L. Jennes et al., Brain Res., 452, 156-164 (1988)] and some kinds of tumors [M. A. Millan et al., Methods Enzymol., 124, 590-606 (1986)] as well as in the pituitaries, and the development of drugs for enhancing or blocking the activity of LH-RH is considered to be effective against various diseases.
For example, leuprorelin acetate which is a highly active derivative of LH-RH (hereinafter referred to as an LH-RH receptor superagonistic compound) [Fujino et al., Biochemical and Biophysical Research Communications, 60, 406-413 (1974); R. T. D. Oliver et al., Br. J. Cancer, 59, 823 (1989); Toguchi et al., J. Int. Med. Res., 18, 35-41 (1990)] reduces release and production of gonadotropic hormones in the pituitaries, induces a decrease in reactivity on gonadotropic hormones in the testes and the ovaries, and inhibits secretion of testosterone and estrogen by repeated administrations. As a result, leuprorelin acetate is known to exhibit antitumor activity on such hormone-dependent cancers such as prostate cancer, and thus has been clinically applied. Further, leuprorelin acetate is also widely clinically used as a therapeutic agent for endometriosis, precocious puberty, etc. The high anticancer activity of leuprorelin acetate is presumed to result from its resistance against proteases, compared with that of natural LH-RH, and high affinity for the LH-RH receptors, which causes desensitization.
However, leuprorelin acetate is the superagonistic compound to the LH-RH receptors, so that transient aggravation accompanied by an increase in serum testosterone concentration due to pituitary-gonadotropic action (acute action) is observed immediately after initial administration. Accordingly, a compound having no superagonistic action, that is an LH-RH receptor antagonistic compound, is expected as a better therapeutic agent for endometriosis, precocious puberty, etc., compared with the LH-RH receptor superagonistic compound.
In general, when a superagonist or an antagonist to a certain biologically active substance's receptor is developed, a compound which has high affinity for a receptor is screened. As the LH-RH receptors, bovine or rat pituitary membrane fractions are now used. However, it is not guaranteed that the screened compounds are effective in humans, because the LH-RH receptors are from different animal species. Human LH-RH receptor cDNA was cloned and the expression thereof in COS7 cells was also reported [S. S. Kakar et al., Biochem. Biophys. Res. Commun., 189, 289-295 (1992)]. However, the expression amount in COS7 cells is extremely small, and further the expression is transient. It is therefore considered that COS7 cells are unsuitable for screening use. Use of human pituitary fractions as human LH-RH receptor protein have been considered. However, human-derived tissues are very difficult to be obtained, resulting-in unsuitableness for screening use.
So, it is desired to develop cells which express a large quantity of human LH-RH receptors in order to screen an LH-RH superagonist or an LH-RH antagonist effectively.
LH-RH is also called GnRH because LH-RH stimulates release of not only LH but also FSH after LH-RH binding to an LH-RH receptor. In the present specification, the term, "LH-RH", is employed.